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They are used for the separation of - large amounts (several grams)
- small amounts (micro- or picogram) of materials
concentration in Solvent B
Stationary Mobile
Stationary Mobile
Adsorption chromatography solid liquid
Paper chromatography semi-solid liquid
Gas chromatography liquid gaseous
Ion-exchange chromatography solid resin liquid
Gel filtration chromatography solid resin liquid
Affinity chromatography solid resin liquid
The mobile and stationary phases are chosen according to the compound to be
separated.
General Techniques of Chromatography
glass
Column Chromatography
All of the major type of chromatographies are carried out using columns:
Adsorption, partition, ion exchange, size exclusion, affinity, gas, high
pressure liquid chromatographies.
Columns: Glass columns are commonly used. Columns should have a support for
the stationary phase.
Peristaltic
pump
Types of elution:
- use single solvent as the eluant
- Gradient elutions, stepwise or continuous, done by changing the
Δ pH
Δ µ (ionic concentration)
Δ polarity
Ex: two solvents having different µ are mixed in the correct proportions.
Buffer+ 0.8 M KCl Buffer (noKCl)
0.8 M--
S2 S1
KCl conc.
(µ) mix
to column 0
If two chambers have the same dimensions (same volume) called linear gradient.
Fraction collection and analysis:
The eluted fractions are collected in separate tubes using an apparatus called
fraction collector.
Works in time
drop or
volume mode
B
A
C
Absorbance
at λmax
Eluants collected in separate tubes using fraction collector. Then each tube is
analyzed for the presence of a desired protein. Ex: total protein
determination and determination of activity of protein due to specific
biological function.
So find that in which tubes (peak or fraction) the protein under interest is eluted.
TLC
It is used for small amount of materials.
glass or plastic
- A slurry of the stationary phase in H2O is applied and coated to a glass, plastic or
foil plate as a uniform thin layer.
- a uniform layer obtained by using a plate spreader starting at one end of
the plate and moving to the other end.
-thickness of the slurry (stationary phase) - 0.25 mm for analytical separations
- 5 mm for preparative separations
- These plates are available commercially.
- Then plates are dried in oven at 100-120°C. This process activates adsorbant
Alumina
Silica adsorbants
Sample Application:
i) Sample is usually low MW molecules
(a.acids, nucleotides, fatty acids, sugars)
solvent front
sample
buffer or solvent mixture (mobile phase)
- Separation done in a glass tank which contains the developing solvent (mobile
phase) to a depth of about 1.5 cm.
- Tank is covered by a glass plate for equilibration with solvent vapor.
- After equilibration, thin layer plate is placed vertically in the tank and covered
again.
- Separation of compounds occurs as the solvents travel up the plate. Polar and non-
polar molecules are separated. Q. How? Principle of separation?
- Development time depends on the solvents - 10-60 minutes
- One of the advantage of TLC, it has a short time for separation.
Solvent system 1
The sample is applied to one corner of a plate as a single spot and plate developed in
one direction in solvent system 1.
Then plate is removed from the tank and dried.
It is developed by another solvent system (system 2) in a direction at right angle to
the first development.
The spot B is further separated into C and D in solvent system 2.
Component Detection:
Qualitative detection methods:
1) Spraying the plate with 50% H2SO4 or 25% H2SO4 in Ethanol
then heating (Δ) burning of compounds and brown spots appear.
2) Examination of the plate under UV light shows the position of UV absorbing
or fluorescent compounds. (use of UV lamp) aromatic compounds
3) Coloured compounds seen with naked eye.
4) Subject the plate to iodine vapor if unsaturated compounds are to be examined.
I
I2|
C=C C-C
|
I
Yellow
5) Spraying the plate with specific colour reagents to develop color. Ex; ninhydrin
for amino acids purple color
6) Autoradiography: used for detection of radioactive compounds. Technique
detects the spots as dark areas on X-ray films.
Quantitative Determination:
The amount of compound may be determined by:
i) On-plate quantification: Quantification of spots still on the plate by
- densitometers: measure the UV or visible absorption of the compound.
- radiochromatogram: used for radio-labelled compounds.
Samp
cut off le
solvent to release the
compound from adsorbent
O Elution
Detection or quantitation by
----------------------------------------------------- colorimetry
Paper Chromatography
Cellulose fibres of paper act as stationary phase (supporting matrix)
may be - polar water
- nonpolar material (liquid paraffin)
Paper Development:
There are two techniques: - ascending
- descending
Paper o
Glass tank
Sample
application o
Solvent ascending
descending
Two dimensional chromatography was also used for paper systems similar to TLC.
Solvent front